Tài liệu Báo cáo khoa học: Compartmentalization and in vivo insulin-induced translocation of the

Compartmentalization and in vivo insulin-induced

translocation of the insulin-signaling inhibitor Grb14

in rat liver

Bernard Desbuquois1,2, Ve´ronique Be´re´ ziat3

, Franc¸ois Authier4

, Jean Girard1,2 and Anne-Franc¸oise

Burnol1,2

1 Institut Cochin, Universite´ Paris Descartes, CNRS (UMR 8104), France

2 Inserm, U567, Paris, France

3 Centre de Recherche Saint-Antoine, UMR S893, Faculte´ de Me´decine Pierre et Marie Curie, Paris, France

4 Inserm, U756, Faculte´ de Pharmacie Paris 11, Chaˆtenay-Malabry, France

Grb14 is a member of the Grb7 ⁄ Grb10 ⁄ Grb14 family

of adaptor proteins, which lack intrinsic enzymatic

activity and share a common multidomain structure.

These adaptors bind to several receptor tyrosine kinases

and signaling proteins, and are involved in the regu￾lation of various processes, including cell growth and

Keywords

endocytosis; insulin receptor; liver;

molecular adaptor; tyrosine kinase activity

Correspondence

B. Desbuquois, De´partement

d’Endocrinologie, Me´tabolisme, Cancer,

Institut Cochin, 24 rue du Faubourg

Saint-Jacques, 75014 Paris, France

Fax: +33 1 44 41 24 21

Tel: +33 1 53 73 27 08

E-mail: [email protected]

(Received 14 September 2007, revised 29

April 2008, accepted 2 July 2008)

doi:10.1111/j.1742-4658.2008.06583.x

The molecular adaptor Grb14 binds in vitro to the activated insulin receptor

(IR) and inhibits IR signaling. In this study, we have used rat liver subcel￾lular fractionation to analyze in vivo insulin effects on Grb14 compartmen￾talization and IR phosphorylation and activity. In control rats, Grb14 was

recovered mainly in microsomal and cytosolic fractions, but was also detect￾able at low levels in plasma membrane and Golgi ⁄ endosome fractions. Insu￾lin injection led to a rapid and dose-dependent increase in Grb14 content,

first in the plasma membrane fraction, and then in the Golgi ⁄ endosome

fraction, which paralleled the increase in IR b-subunit tyrosine phosphory￾lation. Upon sustained in vivo IR tyrosine phosphorylation induced by

high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous

phosphatases, and in vivo phosphorylation of the IR induced by injection of

bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phospha￾tase inhibitor, we observed a striking correlation between IR phosphoryla￾tion state and Grb14 content in both the plasma membrane and

Golgi ⁄ endosome fractions. In addition, coimmunoprecipitation experiments

provided evidence that Grb14 was associated with phosphorylated IR

b-subunit in these fractions. Altogether, these data support a model whereby

insulin stimulates the recruitment of endogenous Grb14 to the activated IR

at the plasma membrane, and induces internalization of the Grb14–IR com￾plex in endosomes. Removal of Grb14 from fractions of insulin-treated rats

by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine

kinase activity, indicating that endogenous Grb14 exerts a negative feedback

control on IR catalytic activity. This study thus demonstrates that Grb14 is

a physiological regulator of liver insulin signaling.

Abbreviations

BPS, between plekstrin homology and SH2; bpV(phen), bisperoxo(1,10-phenanthroline) oxovanadate; EGF, epidermal growth factor; ER,

endoplasmic reticulum; GST, glutathione S-transferase; IR, insulin receptor; IRS-1, insulin receptor substrate-1; IRb, insulin receptor

b-subunit; PDK-1, 3-phosphoinositide-dependent kinase-1; PI3-kinase, phosphoinositide-3-kinase; PIR, phosphorylated insulin receptor￾interacting region; WGA, wheat germ agglutinin.

FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4363